Under-oil open microfluidic system (UOMS) for studying systemic fungal infection

项目摘要/摘要 机会性真菌病原体是医疗保健相关血流感染的主要原因。念珠菌 酵母菌尤其引起80%-90%的与生物膜相关的侵袭性真菌感染和死亡率 接近50%。此外，随着导管和导管的使用增加，念珠菌感染的发生率也在上升 其他基于设备的干预。到目前为止，大多数与真菌感染和潜在的 治疗的重点是生物被膜及其预防。然而，最近的证据表明，这种分散 酵母细胞从生物膜(进入血液)和这些分散的细胞的持久性是 也许是更重要的毒力因素，代表着重要但未得到充分利用的治疗目标。 此外，现有的分析和仪器不适用于测量分散度和表型。 分散的细胞。它们也不概括体内的条件(例如，流动，与宿主细胞的相互作用)。因此，在这里，我们 将开发一种新型的油下开放式微流控系统(UOMS)来量化石油的分散能力 并评估念珠菌突变株和临床分离株中分散细胞的表型。UOMS 平台建立在一种新观察到的现象的基础上，这种现象被称为排他性液体排斥(ELR)。 ELR提供了一种独特的环境，在这种环境中，液体完全从固体表面排斥以消除 生物污垢。此外，ELR扩展了简单开放式微流控设备的功能，使我们能够 克服当前方法的局限性，提供一种能够定量研究真菌的系统 分散。我们将首先(目标1)开发一种油下微通道装置来测量石油的分散能力 念珠菌生物被膜和毒力表型。第二(目标2)我们将使UOMS自动化，并开发一个单一的- 细胞分布试验测量单个分散细胞的表型。最后(目标3)，我们将使用 UOMS平台，开发念珠菌突变体库和临床分离株的分散表型图谱。我们 将测量数百株临床白色念珠菌分散细胞的分散能力和表型 现有文库中可用的分离株和突变体，为分散的遗传决定因素提供线索。

Project Summary/Abstract Opportunistic fungal pathogens are a leading cause of healthcare associated bloodstream infections. Candida yeasts, specifically, cause 80-90% of biofilm-associated invasive fungal infections and mortality rates can approach 50%. Furthermore, the incidence of Candida infections is rising with the increased use of catheter and other device-based interventions. To date, the majority of work related to fungal infections and potential treatments has focused on biofilms and their prevention. However, recent evidence suggests that the dispersion of yeast cells from the biofilm (into the bloodstream) and the persistence of these dispersed cells are perhaps more important virulence factors and represent significant but underutilized treatment targets. Further, existing assays and instrumentation are not amenable to measuring dispersion and phenotyping dispersed cells. Nor do they recapitulate in vivo conditions (e.g. flow, interaction with host cells). Thus, here we will develop a new type of under-oil open microfluidic system (UOMS) to quantify the dispersive capacity of biofilms and assess the phenotype of dispersed cells in Candida mutants and clinical isolates. The UOMS platform is built on the foundation of a newly observed phenomena called Exclusive Liquid Repellency (ELR). ELR provides a unique environment where liquid is completely repelled from a solid surface to eliminate biofouling. Additionally, ELR expands the capabilities of simple open microfluidic devices allowing us to overcome the limitations of current methods and provide a system capable of quantitatively studying fungal dispersion. We will first (Aim 1) develop an under-oil microchannel device to measure the dispersive capacity of Candida biofilms and virulence phenotypes. Second (Aim 2) we will automate the UOMS and develop a single- cell distribution assay to measure the phenotype of individual dispersed cells. And finally (Aim 3), we will use the UOMS platform to develop dispersion phenotype profiles for Candida mutant libraries and clinical isolates. We will measure the dispersive capacity and phenotype of dispersed cells from hundreds of Candida albicans clinical isolates and mutants available in existing libraries, providing clues to the genetic determinants of dispersion.